1. Field of the Invention
This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to the identification of proteins involved in programmed cell death and associations of these proteins.
2. Background Information
Programmed cell death is a physiologic process that ensures homeostasis is maintained between cell production and cell turnover in essentially all self-renewing tissues. In many cases, characteristic morphological changes, termed xe2x80x9capoptosis,xe2x80x9d occur in a dying cell. Since similar changes occur in different types of dying cells, cell death appears to proceed through a common pathway in different cell types.
In addition to maintaining tissue homeostasis, apoptosis also occurs in response to a variety of external stimuli, including growth factor deprivation, alterations in calcium levels, free-radicals, cytotoxic lymphokines, infection by some viruses, radiation and most chemotherapeutic agents. Thus, apoptosis is an inducible event that likely is subject to similar mechanisms of regulation as occur, for example, in a metabolic pathway. In this regard, dysregulation of apoptosis also can occur and is observed, for example, in some types of cancer cells, which survive for a longer time than corresponding normal cells, and in neurodegenerative diseases where neurons die prematurely. In viral infections, induction of apoptosis can figure prominently in the pathophysiology of the disease process, because immune-based eradication of viral infections depends on elimination of virus-producing host cells by immune cell attack resulting in apoptosis.
Some of the proteins involved in programmed cell death have been identified and associations among some of these proteins have been described. However, additional apoptosis regulating proteins remain to be found and the mechanisms by which these proteins mediate their activity remains to be elucidated. The identification of the proteins involved in cell death and an understanding of the associations between these proteins can provide a means for manipulating the process of apoptosis in a cell and, therefore, selectively regulating the relative lifespan of a cell or its relative resistance to cell death stimuli.
The principal effectors of apoptosis are a family of intracellular proteases known as Caspases, representing an abbreviation for Cysteine Aspartyl Proteases. Caspases are found as inactive zymogens in essentially all animal cells. During apoptosis, the caspases are activated by proteolytic processing at specific aspartic acid residues, resulting in the production of subunits that assemble into an active protease typically consisting of a heterotetramer containing two large and two small subunits (Thornberry and Lazebnik, Science 281:1312-1316 (1998)). The phenomenon of apoptosis is produced directly or indirectly by the activation of caspases in cells, resulting in the proteolytic cleavage of specific substrate proteins. Moreover, in many cases, caspases can cleave and activate themselves and each other, creating cascades of protease activation and mechanisms for xe2x80x9cautoxe2x80x9d-activation.
Among the substrates of caspases are the intracellular proforms of cytokines such as pro-Interleukin-1xcex2 (pro-IL-1xcex2) and pro-IL-18. When cleaved by caspases, these pro-proteins are converted to the biologically active cytokines which are then liberated from cells, circulating in the body and eliciting inflammatory immune reactions. Thus, caspases can be involved, in some instances, in cytokine activation and responses to infectious agents, as well as inflammatory and autoimmune diseases. Caspases also participate in signal transduction pathways activated by some cytokine receptors, particularly members of the Tumor Necrosis Factor (TNF) family of cytokine receptors which are capable of activating certain caspase zymogens.
Thus, knowledge about the proteins having domains that interact with and regulate caspases is important for devising strategies for manipulating cell life and death in therapeutically useful ways. The identification of such proteins that contain caspase-interacting domains and the elucidation of the proteins with which they interact, therefore, can form the basis for strategies designed to modulate apoptosis, cytokine production, cytokine receptor signaling, and other cellular processes. Thus a need exists to identify proteins that interact with caspases and other apoptosis related proteins. The present invention satisfies this need and provides additional advantages as well.
In accordance with the present invention, there are provided novel xe2x80x9cNB-ARC and CARDxe2x80x9d-containing proteins, designated NAC, as well as several isoforms of NAC produced by alternative mRNA splicing. The invention also provides nucleic acid molecules encoding NAC and its isoforms, vectors containing these nucleic acid molecules and host cells containing the vectors. The invention also provides antibodies that can specifically bind to NAC proteins, including alternative isoforms thereof.
The present invention also provides a screening assay useful for identifying agents that can effectively alter the association of NAC with itself or with other proteins. By altering the self-association of NAC or by altering their interactions with other proteins, an effective agent may increase or decrease the level of caspase proteolytic activity or apoptosis in a cell, or it may increase or decrease the levels of NF-xcexaB, cytokine production, or other events.
The invention also provides methods of altering the activity of NAC in a cell, wherein such increased or decreased activity of NAC can modulate the level of apoptosis or other cellular responses. For example, the activity of NAC in a cell can be increased by introducing into the cell and expressing a nucleic acid sequence encoding these proteins. In addition, the activity of NAC in a cell can be decreased by introducing into the cell and expressing a fragment of NAC, or an antisense nucleotide sequence that is complementary to a portion of a nucleic acid molecule encoding the NAC proteins.
The invention also provides methods for using an agent that can specifically bind NAC or a nucleotide sequence that can bind to a nucleic acid molecule encoding NAC to diagnose a pathology that is characterized by an altered level of apoptosis due to an increased or decreased level of NAC in a cell.